Chapter 10: Disorders of Sex Development

Sex development begins in utero but continues into young adulthood with the achievement of sexual maturity and reproductive capability. The major determinants of sex development can be divided into three components: chromosomal sex, gonadal sex (sex determination), and phenotypic sex (sex differentiation) (Fig. 10-1). Variations at each of these stages can result in disorders (or differences) of sex development (DSDs) (Table 10-1). In the newborn period, approximately 1 in 4000 babies require investigation because of ambiguous (atypical) genitalia. Urgent assessment is required, because some causes such as congenital adrenal hyperplasia (CAH) can be associated with life-threatening adrenal crises. Support for the parents and clear communication about the diagnosis and management options are essential. The involvement of an experienced multidisciplinary team is important for counseling, planning appropriate investigations, and discussing long-term well-being. DSDs can also present at other ages and to a range of health professionals. Subtler forms of gonadal dysfunction (e.g., Klinefelter’s syndrome [KS], Turner’s syndrome [TS]) often are diagnosed later in life by internists. Because these conditions are associated with a variety of psychological, reproductive, and potential medical consequences, an open dialogue must be established between the patient and health care providers to ensure continuity and attention to these issues.

Chromosomal sex, defined by a karyotype, describes the X and/or Y chromosome complement (46,XY; 46,XX) that is established at the time of fertilization. The presence of a normal Y chromosome determines that testis development will occur even in the presence of multiple X chromosomes (e.g., 47,XXY or 48,XXXY). The loss of an X chromosome impairs gonad development (45,X or 45,X/46,XY mosaicism). Fetuses with no X chromosome (45,Y) are not viable.

Gonadal sex refers to the histologic and functional characteristics of gonadal tissue as testis or ovary. The embryonic gonad is bipotential and can develop (from ~42 days after conception) into either a testis or an ovary, depending on which genes are expressed (Fig. 10-2). Testis development is initiated by expression of the Y chromosome gene SRY (sex-determining region on the Y chromosome) that encodes an HMG box transcription factor. SRY is expressed transiently in cells destined to become Sertoli cells and serves as a pivotal switch to establish the testis lineage. Mutation of SRY prevents testis development in 46,XY individuals, whereas translocation of SRY in 46,XX individuals is sufficient to induce testis development and a male phenotype. Other genes are necessary to continue testis development. SOX9 (SRY-related HMG-box gene 9) is upregulated by SRY in the developing testis but is suppressed in the ovary. WT1 (Wilms’ tumor–related gene 1) acts early in the genetic pathway and regulates the transcription of several genes, including SFI (NR5A1), DAX1 (NR0B1), and AMH (encoding müllerian-inhibiting substance [MIS]). SF1 encodes steroidogenic factor 1, a nuclear receptor that functions in cooperation with other transcription factors to regulate a large array of adrenal and gonadal genes, including SOX9 and many genes involved in steroidogenesis. SF1 mutations causing loss of function are found in ~10% of XY patients with gonadal dysgenesis and impaired androgenization. In contrast, duplication of a related gene DAX1 also impairs testis development, revealing the exquisite sensitivity of the testis-determining pathway to gene dosage effects. DAX1 loss-of-function mutations cause adrenal hypoplasia, hypogonadotropic hypogonadism, and testicular dysgenesis. In addition to the genes mentioned above, studies of humans and mice indicate that at least 30 other genes are also involved in gonad development (Fig.  10-2). These genes encode an array of signaling molecules and paracrine growth factors in addition to transcription factors.

Although ovarian development once was considered a “default” process, it is now clear that specific genes are expressed during the earliest stages of ovary development. Some of these factors may repress testis development (e.g., WNT4, R-spondin-1) (Fig.  10-2). Once the ovary has formed, additional factors are required for normal follicular development (e.g., follicle-stimulating hormone [FSH] receptor, GDF9). Steroidogenesis in the ovary requires the development of follicles that contain granulosa cells and theca cells surrounding the oocytes (Chap. 13). Thus, there is relatively limited ovarian steroidogenesis until puberty.

Germ cells also develop in a sex dimorphic manner. In the developing ovary, primordial germ cells (PGCs) proliferate and enter meiosis, whereas they proliferate and then undergo mitotic arrest in the developing testis. PGC entry into meiosis is initiated by retinoic acid that activates STRA8 (stimulated by retinoic acid 8) and other genes involved in meiosis. The developing testis produces high levels of CYP26B1, an enzyme that degrades retinoic acid, preventing PGC entry into meiosis. Approximately 7 million germ cells are present in the fetal ovary in the second trimester, and 1 million remain at birth. Only 400 are ovulated during a woman’s reproductive life span (Chap. 13).

Phenotypic sex refers to the structures of the external and internal genitalia and secondary sex characteristics. The developing testis releases anti-müllerian hormone (AMH; also known as müllerian-inhibiting substance [MIS]) from Sertoli cells and testosterone from Leydig cells. AMH is a member of the transforming growth factor (TGF) β family and acts through specific receptors to cause regression of the müllerian structures from 60–80 days after conception. At ~60–140 days after conception, testosterone supports the development of wolffian structures, including the epididymides, vasa deferentia, and seminal vesicles. Testosterone is the precursor for dihydrotestosterone (DHT), a potent androgen that promotes development of the external genitalia, including the penis and scrotum (65–100 days, and thereafter) (Fig. 10-3). The urogenital sinus develops into the prostate and prostatic urethra in the male and into the urethra and lower portion of the vagina in the female. The genital tubercle becomes the glans penis in the male and the clitoris in the female. The urogenital swellings form the scrotum or the labia majora, and the urethral folds fuse to form the shaft of the penis and the male urethra or the labia minora. In the female, wolffian ducts regress and the müllerian ducts form the fallopian tubes, uterus, and upper segment of the vagina. A female phenotype will develop in the absence of the gonad, but estrogen is needed for maturation of the uterus and breast at puberty.

Variations in sex chromosome number and structure can present as DSDs (e.g., 45,X/46,XY). KS (47,XXY) and TS (45,X) do not usually present with genital ambiguity but are associated with gonadal dysfunction (Table 10-2).

KLINEFELTER’S SYNDROME (47,XXY)

Pathophysiology

The classic form of KS (47,XXY) occurs after meiotic nondisjunction of the sex chromosomes during gametogenesis (40% during spermatogenesis, 60% during oogenesis). Mosaic forms of KS (46,XY/47,XXY) are thought to result from chromosomal mitotic nondisjunction within the zygote and occur in at least 10% of individuals with this condition. Other chromosomal variants of KS (e.g., 48,XXYY, 48,XXXY) have been reported but are less common.

Clinical features

KS is characterized by small testes, infertility, gynecomastia, tall stature/increased leg length, and hypogonadism in phenotypic males. It has an incidence of at least 1 in 1000 men, but approximately 75% of cases are not diagnosed. Of those who are diagnosed, only 10% are identified prepubertally, usually because of small genitalia or cryptorchidism. Others are diagnosed after puberty, usually based on impaired androgenization and/or gynecomastia. Developmental delay, speech difficulties, and poor motor skills may be features but are variable, especially in adolescence. Later in life, body habitus or infertility leads to the diagnosis. Testes are small and firm (median length 2.5 cm [4 mL volume]; almost always <3.5 cm [12 mL]) and typically seem inappropriately small for the degree of androgenization. Biopsies are not usually necessary but typically reveal seminiferous tubule hyalinization and azoospermia. Other clinical features of KS are listed in Table 10-2. Plasma concentrations of FSH and luteinizing hormone (LH) are increased in most adults with 47,XXY, and plasma testosterone is decreased (50–75%), reflecting primary gonadal failure. Estradiol is often increased, likely because of chronic Leydig cell stimulation by LH and aromatization of androstenedione by adipose tissue; the increased ratio of estradiol-to-testosterone results in gynecomastia (Chap. 11). Patients with mosaic forms of KS have less severe clinical features, have larger testes, and sometimes achieve spontaneous fertility.

TREATMENT

TURNER’S SYNDROME (GONADAL DYSGENESIS; 45,X)

Pathophysiology

Approximately one-half of women with TS have a 45,X karyotype, about 20% have 45,X/46,XX mosaicism, and the remainder have structural abnormalities of the X chromosome such as X fragments, isochromosomes, or rings. The clinical features of TS result from haploinsufficiency of multiple X chromosomal genes (e.g., short stature homeobox, SHOX). However, imprinted genes also may be affected when the inherited X has different parental origins.

Clinical features

TS is characterized by bilateral streak gonads, primary amenorrhea, short stature, and multiple congenital anomalies in phenotypic females. It affects ~1 in 2500 women and is diagnosed at different ages depending on the dominant clinical features (Table 10-2). Prenatally, a diagnosis of TS usually is made incidentally after chorionic villus sampling or amniocentesis for unrelated reasons such as advanced maternal age. Prenatal ultrasound findings include increased nuchal translucency. The postnatal diagnosis of TS should be considered in female neonates or infants with lymphedema, nuchal folds, low hairline, or left-sided cardiac defects and in girls with unexplained growth failure or pubertal delay. Although limited spontaneous pubertal development occurs in up to 30% of girls with TS (10%, 45,X; 30–40%, 45,X/46,XX) and ~2% reach menarche, the vast majority of women with TS develop complete ovarian insufficiency. Therefore, this diagnosis should be considered in all women who present with primary or secondary amenorrhea and elevated gonadotropin levels.

TREATMENT

45,X/46,XY MOSAICISM (MIXED GONADAL DYSGENESIS)

The phenotype of individuals with 45,X/46,XY mosaicism (sometimes called mixed gonadal dysgenesis) can vary considerably. Some have a predominantly female phenotype with somatic features of TS, streak gonads, and müllerian structures, and are managed as TS with a Y chromosome. Most 45,X/46,XY individuals have a male phenotype and testes, and the diagnosis is made incidentally after amniocentesis or during investigation of infertility. In practice, most newborns referred for assessment have atypical genitalia and variable somatic features. Management is complex and needs to be individualized. A female sex-of-rearing is often assigned if uterine structures are present, gonads are intraabdominal, and phallic development is incomplete. In such situations, gonadectomy usually is considered to prevent further androgen secretion at puberty and prevent risk of gonadoblastoma (up to 25%). Individuals raised as males usually require reconstructive surgery for hypospadias and removal of dysgenetic or streak gonads if the gonads cannot be brought down into the scrotum. Scrotal testes can be preserved but require regular examination for tumor development and sonography at the time of puberty. Biopsy for carcinoma in situ is recommended in adolescence, and testosterone supplementation may be required to support androgenization in puberty or if low testosterone is detected in adulthood. Height potential is usually attenuated; some children receive recombinant growth hormone using TS protocols. Screening for cardiac, renal, and other TS features should be considered, and psychological support offered for the family and young person.

OVOTESTICULAR DSD

Ovotesticular DSD (formerly called true hermaphroditism) occurs when both an ovary and a testis—or when an ovotestis—are found in one individual. Most individuals with this diagnosis have a 46,XX karyotype, especially in sub-Saharan Africa, and present with ambiguous genitalia at birth or with breast development and phallic development at puberty. A 46,XX/46,XY chimeric karyotype is less common and has a variable phenotype.

Disorders of gonadal and phenotypic sex can result in underandrogenization of individuals with a 46,XY karyotype (46,XY DSD) and the excess androgenization of individuals with a 46,XX karyotype (46,XX DSD) (Table 10-1). These disorders cover a spectrum of phenotypes ranging from “46,XY phenotypic females” or “46,XX phenotypic males” to individuals with atypical genitalia.

46,XY DSD

Underandrogenization of the 46,XY fetus (formerly called male pseudohermaphroditism) reflects defects in androgen production or action. It can result from disorders of testis development, defects of androgen synthesis, or resistance to testosterone and DHT (Table 10-1).

Disorders of testis development 

Testicular dysgenesis

Pure (or complete) gonadal dysgenesis (Swyer’s syndrome) is associated with streak gonads, müllerian structures (due to insufficient AMH/MIS secretion), and a complete absence of androgenization. Phenotypic females with this condition often present because of absent pubertal development and are found to have a 46,XY karyotype. Serum sex steroids, AMH/MIS, and inhibin B are low, and LH and FSH are elevated. Patients with partial gonadal dysgenesis (dysgenetic testes) may produce enough MIS to regress the uterus and sufficient testosterone for partial androgenization, and therefore usually present in the newborn period with atypical genitalia. Gonadal dysgenesis can result from mutations or deletions of testis-promoting genes (WT1, CBX2, SF1, SRY, SOX9, MAP3K1, DHH, GATA4, ATRX, ARX, DMRT) or duplication of chromosomal loci containing “antitestis” genes (e.g., WNT4/RSPO1, DAX1) (Table 10-3). Among these, deletions or mutations of SRY and heterozygous mutations of SF1 (NR5A1) appear to be most common but still account collectively for <25% of cases. Associated clinical features may be present, reflecting additional functional roles for these genes. For example, renal dysfunction occurs in patients with specific WT1 mutations (Denys-Drash and Frasier’s syndromes), primary adrenal failure occurs in some patients with SF1 mutations, and severe cartilage abnormalities (campomelic dysplasia) are the predominant clinical feature of SOX9 mutations. A family history of DSD, infertility, or early menopause is important because mutations in SF1/NR5A1 can be inherited from a mother in a sex-limited dominant manner (which can mimic X-linked inheritance). In some cases, a woman may later develop primary ovarian insufficiency because of the effect of SF1 on the ovary. Intraabdominal dysgenetic testes should be removed to prevent malignancy, and estrogens can be used to induce secondary sex characteristics and uterine development in 46,XY individuals raised as females, if it is felt that a female gender identity is established. Absent (vanishing) testis syndrome (bilateral anorchia) reflects regression of the testis during development. The etiology is unknown, but the absence of müllerian structures indicates adequate secretion of AMH early in utero. In most cases, androgenization of the external genitalia is either normal or slightly impaired (e.g., small penis, hypospadias). These individuals can be offered testicular prostheses and should receive androgen replacement in adolescence.

Disorders of androgen synthesis

Defects in the pathway that regulates androgen synthesis (Fig. 10-4) cause underandrogenization of the 46,XY fetus (Table 10-1). Müllerian regression is unaffected because Sertoli cell function is preserved. Most of these conditions can present with a spectrum of genital phenotypes, ranging from female-typical external genitalia or clitoromegaly in the more severe situations to penoscrotal hypospadias or a small phallus in others.

LH receptor

Mutations in the LH receptor (LHCGR) cause Leydig cell hypoplasia and androgen deficiency, due to impaired actions of human chorionic gonadotropin in utero and LH late in gestation and during the neonatal period. As a result, testosterone and DHT synthesis are insufficient for complete androgenization.

Steroidogenic enzyme pathways

Mutations in steroidogenic acute regulatory protein (StAR) and CYP11A1 affect both adrenal and gonadal steroidogenesis (Fig.  10-4) (Chap. 8). Affected individuals (46,XY) usually have severe early-onset salt-losing adrenal failure and a female phenotype, although later-onset milder variants have been reported. Defects in 3β-hydroxysteroid dehydrogenase type 2 (HSD3β2) also cause adrenal insufficiency in severe cases, but the accumulation of dehydroepiandrosterone (DHEA) has a mild androgenizing effect, resulting in ambiguous genitalia or hypospadias. Salt loss occurs in many but not all cases. Patients with CAH due to 17α-hydroxylase (CYP17) deficiency have variable underandrogenization and develop hypertension and hypokalemia due to the potent salt-retaining effects of corticosterone and 11-deoxycorticosterone. Patients with complete loss of 17α-hydroxylase function often present as phenotypic females who fail to enter puberty and are found to have inguinal testes and hypertension in adolescence. Some mutations in CYP17 selectively impair 17,20-lyase activity without altering 17α-hydroxylase activity, leading to underandrogenization without mineralocorticoid excess and hypertension. Disruption of the coenzyme, cytochrome b5 (CYB5A), can present similarly, and methemoglobinemia is usually present. Mutations in P450 oxidoreductase (POR) affect multiple steroidogenic enzymes, leading to impaired androgenization and a biochemical pattern of apparent combined 21-hydroxylase and 17α-hydroxylase deficiency, sometimes with skeletal abnormalities (Antley-Bixler craniosynostosis). Defects in 17β-hydroxysteroid dehydrogenase type 3 (HSD17β3) and 5α-reductase type 2 (SRD5A2) interfere with the synthesis of testosterone and DHT, respectively. These conditions are characterized by minimal or absent androgenization in utero, but some phallic development can occur during adolescence due to the action of other enzyme isoforms. Individuals with 5α-reductase type 2 deficiency have normal wolffian structures and usually do not develop breast tissue. At puberty, the increase in testosterone induces muscle mass and other virilizing features despite DHT deficiency. Some individuals change gender from female to male at puberty. Thus, the management of this disorder is challenging. DHT cream can improve prepubertal phallic growth in patients raised as male. Gonadectomy before adolescence and estrogen replacement at puberty can be considered in individuals raised as females who have a female gender identity. Disruption of alternative pathways to fetal DHT production might also present with 46,XY DSD (AKR1C2/AKR1C4).

Disorders of androgen action

Androgen insensitivity syndrome

Mutations in the androgen receptor cause resistance to androgen (testosterone, DHT) action or the androgen insensitivity syndrome (AIS). AIS is a spectrum of disorders that affects at least 1 in 100,000 46,XY individuals. Because the androgen receptor is X-linked, only 46,XY offspring are affected if the mother is a carrier of a mutation. XY individuals with complete AIS (formerly called testicular feminization syndrome) have a female phenotype, normal breast development (due to aromatization of testosterone), a short vagina but no uterus (because MIS production is normal), scanty pubic and axillary hair, and a female gender identity and sex role behavior. Gonadotropins and testosterone levels can be low, normal, or elevated, depending on the degree of androgen resistance and the contribution of estradiol to feedback inhibition of the hypothalamic-pituitary-gonadal axis. AMH/MIS levels in childhood are normal or high. Most patients present with inguinal hernias (containing testes) in childhood or with primary amenorrhea in late adolescence. Gonadectomy sometimes is offered for girls diagnosed in childhood, because there is a low risk of malignancy, and estrogen replacement is prescribed. Alternatively, the gonads can be left in situ until breast development is complete and removed because of tumor risk. Some adults with complete AIS decline gonadectomy, but should be counseled about the risk of malignancy, especially because early detection of premalignant changes by imaging or biomarkers is currently not possible. The use of graded dilators in adolescence is usually sufficient to dilate the vagina for sexual intercourse.

Partial AIS (Reifenstein’s syndrome) results from androgen receptor mutations that maintain residual function. Patients often present in infancy with penoscrotal hypospadias and small undescended testes and with gynecomastia at the time of puberty. Those individuals raised as males usually require hypospadias repair in childhood and may need breast reduction in adolescence. Some boys enter puberty spontaneously. High-dose testosterone has been given to support development if puberty does not progress, but long-term data are limited. More severely underandrogenized patients present with clitoral enlargement and labial fusion and may be raised as females. The surgical and psychosexual management of these patients is complex and requires active involvement of the parents and the patient during the appropriate stages of development. Azoospermia and male-factor infertility also have been described in association with mild loss-of-function mutations in the androgen receptor.

OTHER DISORDERS AFFECTING 46,XY MALES

Persistent müllerian duct syndrome is the presence of a uterus in an otherwise phenotypic male. This condition can result from mutations in AMH or its receptor (AMHR2). The uterus may be removed, but only if damage to the vasa deferentia and blood supply can be avoided. Isolated hypospadias occurs in ~1 in 250 males and is usually repaired surgically. Most cases are idiopathic, although evidence of penoscrotal hypospadias, poor phallic development, and/or bilateral cryptorchidism requires investigation for an underlying DSD (e.g., partial gonadal dysgenesis, mild defect in testosterone action, or even severe forms of 46,XX CAH). Unilateral undescended testes (cryptorchidism) affect more than 3% of boys at birth. Orchidopexy should be considered if the testis has not descended by 6–9 months of age. Bilateral cryptorchidism occurs less frequently and should raise suspicion of gonadotropin deficiency or DSD. A small subset of patients with cryptorchidism may have mutations in the insulin-like 3 (INSL3) gene or its receptor LGR8 (also known as GREAT), which mediates normal testicular descent. Syndromic associations and intrauterine growth retardation also occur relatively frequently in association with impaired testicular function or target tissue responsiveness, but the underlying etiology of many of these conditions is unknown.

46,XX DSD

Inappropriate androgenization of the 46,XX fetus (formerly called female pseudohermaphroditism) occurs when the gonad (ovary) contains androgen-secreting testicular material or after increased androgen exposure, which is usually adrenal in origin (Table 10-1).

46,XX testicular/ovotesticular DSD

Testicular tissue can develop in 46,XX testicular DSD (46,XX males) after translocation of SRY, duplication of SOX9, or defects in RSPO1 (Table 10-4).

Increased androgen exposure

21-Hydroxylase deficiency (congenital adrenal hyperplasia)

The classic form of 21-hydroxylase deficiency (21-OHD) is the most common cause of CAH (Chap. 8). It has an incidence between 1 in 10,000 and 1 in 15,000 and is the most common cause of androgenization in chromosomal 46,XX females (Table 10-4). Affected individuals are homozygous or compound heterozygous for severe mutations in the enzyme 21-hydroxylase (CYP21A2). This mutation causes a block in adrenal glucocorticoid and mineralocorticoid synthesis, increasing 17-hydroxyprogesterone and shunting steroid precursors into the androgen synthesis pathway (Fig.  10-4). Glucocorticoid insufficiency causes a compensatory elevation of adrenocorticotropin (ACTH), resulting in adrenal hyperplasia and additional synthesis of steroid precursors proximal to the enzymatic block. Increased androgen synthesis in utero causes androgenization of the 46,XX fetus in the first trimester. Ambiguous genitalia are seen at birth, with varying degrees of clitoral enlargement and labial fusion. Excess androgen production causes gonadotropin-independent precocious puberty in males with 21-OHD.

The salt-wasting form of 21-OHD results from severe combined glucocorticoid and mineralocorticoid deficiency. A salt-wasting crisis usually manifests between 5 and 21 days of life and is a potentially life-threatening event that requires urgent fluid resuscitation and steroid treatment. Thus, a diagnosis of 21-OHD should be considered in any baby with atypical genitalia with bilateral nonpalpable gonads. Males (46,XY) with 21-OHD have no genital abnormalities at birth but are equally susceptible to adrenal insufficiency and salt-losing crises.

Females with the classic simple virilizing form of 21-OHD also present with genital ambiguity. They have impaired cortisol biosynthesis but do not develop salt loss. Patients with nonclassic 21-OHD produce normal amounts of cortisol and aldosterone but at the expense of producing excess androgens. Hirsutism (60%), oligomenorrhea (50%), and acne (30%) are the most common presenting features. This is one of the most common recessive disorders in humans, with an incidence as high as 1 in 100 to 500 in many populations and 1 in 27 in Ashkenazi Jews of Eastern European origin.

Biochemical features of acute salt-wasting 21-OHD are hyponatremia, hyperkalemia, hypoglycemia, inappropriately low cortisol and aldosterone, and elevated 17-hydroxyprogesterone, ACTH, and plasma renin activity. Presymptomatic diagnosis of classic 21-OHD is now made by neonatal screening tests for increased 17-hydroxyprogesterone in many centers. In most cases, 17-hydroxyprogesterone is markedly increased. In adults, ACTH stimulation (0.25 mg of cosyntropin IV) with assays for 17-hydroxyprogesterone at 0 and 30 min can be useful for detecting nonclassic 21-OHD and heterozygotes (Chap. 8).

TREATMENT

Other causes

Increased androgen synthesis can also occur in CAH due to defects in POR, 11β-hydroxylase (CYP11B1), and 3β-hydroxysteroid dehydrogenase type 2 (HSD3B2) and with mutations in the genes encoding aromatase (CYP19) and the glucocorticoid receptor. Increased androgen exposure in utero can occur with maternal virilizing tumors and with ingestion of androgenic compounds.

OTHER DISORDERS AFFECTING 46,XX FEMALES

Congenital absence of the vagina occurs in association with müllerian agenesis or hypoplasia as part of the Mayer-Rokitansky-Kuster-Hauser (MRKH) syndrome (rarely caused by WNT4 mutations). This diagnosis should be considered in otherwise phenotypically normal females with primary amenorrhea. Associated features include renal (agenesis) and cervical spinal abnormalities.

GLOBAL CONSIDERATIONS

The approach to a child or adolescent with ambiguous genitalia or another DSD requires cultural sensitivity, as the concepts of sex and gender vary widely. Rare genetic DSDs can occur more frequently in specific populations (e.g., 5α-reductase type 2 in the Dominican Republic). Different forms of CAH also show ethnic and geographic variability. In many countries, appropriate biochemical tests may not be readily available, and access to appropriate forms of treatment and support may be limited.